Salmonellosis, due to non-typhoid Salmonella enterica serovars, is one of the most frequently reported food-borne diseases worldwide. Since 1993 to 2003, Enteritidis (84%), Typhimurium (7%), Virchow (1%), Infantis (1%) and Blockley (1%), were the serovars of S. enterica mainly isolated from human salmonellosis in European Union. More recently, data from 2006 to 2007 corroborate that Enteritidis and Typhimurium continue to be the most prevalent, although an increase of Typhimurium (16.5%) and a decrease of Enteritidis (64.5%) must be noted (EFSA, 2010).
Poultry and swine are known reservoirs of these zoonotic pathogens. S. Enteritidis is the most frequent cause of human salmonellosis at European Community level. In general, this serovar is also the most frequently isolated from poultry meat and especially in table eggs, whereas it is less commonly found from pigs and cattle and products thereof. The second most prevalent serovar in humans, S. Typhimurium, is the most frequently isolated serovar in pigs, cattle and products thereof and was also among the top three serovars isolated from broilers and table eggs (EFSA, 2010). It is also well documented that in the last decade, the antibiotic resistance is increasing in non-typhoid S. enterica, which means that animals are both pathogen and resistance reservoirs.
Due to the great concern about Salmonella zoonoses, a new Salmonella control program in breeding flocks of Gallus gallus was implemented. This control program aim to meet the Salmonella reduction targets set by the Regulation (EC) No 1003/2005 and covers the following serovars: S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow and S. Hadar. Likewise, a control or monitoring program for Salmonella in pig herds or slaughter pigs have been implemented in different countries with the aim to have more robust data about Salmonella prevalence in swine production. Different strategies and measures to reduce Salmonella have been proposed. In fact, the intensified control of Salmonella in animal populations, particularly in poultry, and a better hygiene throughout the food chain should be the cause of the decrease of the notification rate of Salmonella cases in the European Union. One of these measures has been the use of life or inactivated vaccines to control S. Enteritidis in poultry. However, some authors doubt of its long term effectiveness (Baumler et al., 2000; Rabsch et al., 2000). In this context, it is necessary to open the scope for further research to explore new approaches to existing ones, and new weapons with which to combat these zoonotic pathogens.
Within this aim, the potential use of bacteriophages in human and animal therapy has been extensively reviewed in these lasts years (Barrow and Soothil, 1997; Barrow, 2001; Sulakvelidze et al., 2001; Summers, 2001; Merrill et al., 2003; Matsuzaki et al., 2005; Parisien et al., 2008;). Moreover, its interest as biocontrol agents of zoonotic bacteria in food and animal production and also of others involved in biodeterioration has also arisen and some strategies have been proposed (Barrow and Soothil, 1997; Joerger, 2002; Callaway et al., 2004; Greer, 2005; Hudson et al., 2005). The special characteristics of phages among which are its high specificity and its ability to selfreplicate when infecting, make them especially attractive to prevent food-borne illness, by using them as biocontrol agents in poultry and swine industries and in food production. However, the narrow host-range of bacteriophages is a great restriction to find good candidate phages for bacteriophage therapy against Salmonella enterica because these phages must be able to infect a wide range of strains and serovars of this bacterial species.
There is a consensus about lytic bacteriophages are the best suitable to achieve a significant reduction of bacterial populations due its rapid bacterial killing. Furthermore, safety criteria related to the spread of virulence genes or antibiotic resistance that could be in the phage genome must also be taken into account. Therefore, it is necessary to obtain genomic data demonstrating the impossibility of a lysogenic cycle and the absence of both potential virulence factors and/or antimicrobial resistance genes in the genomes of phage for therapy applications. In addition, it must be noted that the effectiveness of phages in food is likely to vary with each phage, each food matrix, and with the conditions of application including environmental factors (EFSA, 2009).
There are some works testing the effect of different bacteriophages for their inhibitory effect against Salmonella enterica. Thus, it has been reported a reduction of S. Typhimurium in the digestive tract of chickens after oral inoculation of lytic bacteriophages, although high titter of bacteriophages were needed and the inoculation was performed soon after infection to maximized the effect (Berchieri et al., 1991). More recently, much more significant decreases in S. Enteritidis in the cecal contents of broilers orally inoculated with bacteriophages, some of them belonging to the Myoviridae and Siphoviridae family, have been obtained at different times (Fiorentin et al., 2005a; Atterbury et al., 2007; Filho et al., 2007; Borie et al., 2008) and also in S. Typhimurium in experiments that combine the oral inoculation of bacteriophages with competitive exclusion cultures (Toro et al., 2005). In another field of application which included food safety, treatment with bacteriophages also reduced the concentration of S. Enteritidis in poultry products and turkey (Goode et al., 2003; Higgins et al., 2005; Fiorentin et al. 2005b; Bigwood et al., 2008). However, all these works are partial studies because any of them show nor a morphological and genomic characterization of bacteriophages, the phage host range, the efficiency of bacteriophages for reducing Salmonella in poultry over long periods and its efficiency in different matrices and neither some criteria related to safety.
The present invention describes the novel lytic bacteriophages UAB_Phi20 and UAB_Phi78, belonging to the Podoviriade family and UAB_Phi87, belonging to the Myoviridae family, all of them of the Caudovirales order. Those bacteriophages are Salmonella specific bacteriophages that infect different serovars of Salmonella and a high percentage of clonally unrelated strains of the serovars Typhimurium and Enteritidis. Those bacteriophages maintain their infective ability at a wide pH range. The genome of those bacteriophages does not include any known or similar gene to those involved in bacterial virulence.
The present invention also describes bacteriophage cocktail compositions containing at least one of the bacteriophages and/or parts and/or products of the bacteriophages of the invention. Those cocktail compositions can also contain other lytic bacteriophages and/or parts and/or products of them. The novel bacteriophages, when administered to animals, are useful for the bacteriophage therapy over the time of different Salmonella serovars. The present invention refers to the use of these novel bacteriophages and the novel bacteriophage cocktail compositions to reduce the concentration of different Salmonella serovars, providing doses and schedule of administration in animals and in different matrices. The invention refers to the use of the novel bacteriophages and the novel safe phage cocktail compositions as an antimicrobial for the biocontrol of populations of Salmonella in animal therapy and livestock in general as well as a sanitization and sterilization composition to be applied on slaughterhouses, animal transportation, food processing industries and foods.